PROJECT SUMMARY Otitis media (OM), or middle ear infection, is a highly prevalent pediatric disease worldwide. It ranks first in reasons why children visit physician's offices and emergency departments, undergo surgery under general anesthesia, and experience hearing loss. Three bacterial species predominate in OM, however due to broad use of capsular conjugate vaccines for Streptococcus pneumoniae (Spn), the microbiology of OM has shifted worldwide, with the relative proportion of cases due to nontypeable Haemophilus influenzae (NTHI) increasing significantly. NTHI are now considered equivalent to Spn as a causative agent of acute OM, yet remain the predominant pathogen of chronic OM and recurrent OM. There is thus a pressing need to develop better methods to manage NTHI-induced OM, preferably via development of vaccines to prevent it. To do so requires a complete understanding of the pathogenesis of disease and precise definition of the immunological target(s) that NTHI present. It is thus important to delineate the role of NTHI phase variation in pathobiology, including adherence, colonization of the nasopharynx (NP), biofilm formation, resistance to effectors of innate and acquired immunity and frequency/severity of induced OM. To date, using both multiple pediatric strain collections and a chinchilla model, we've shown that the NTHI ModA2 phasevarion is active as NTHI transition from benign colonization of the NP to overt infection of the middle ear and further, that it plays a major role in pathogenesis of experimental OM and altered expression of surface/vaccine antigens. Despite these intriguing observations, there remain multiple gaps in our understanding of NTHI phasevarions, the impact of epigenetic changes on pathogenesis, and the effect of phasevarion-mediated regulation of gene expression on selection of optimal vaccine candidates. Thereby, we propose two highly complementary and integrated specific aims to fill these gaps. In Specific Aim 1, strong preliminary data on differential gene regulation in modA2, 4, 5, 9 & 10 phasevarions will support the conduct of a series of in vitro studies designed to probe the known niche-specific conditions that combine with phasevarion-mediated methylation changes to alter gene expression in NTHI. Aim 1 studies are designed to deconstruct the multifactorial in vivo environment in order to individually characterize key biological selective pressures that act on phasevarion switching. In Specific Aim 2, studies of modA4, 5, 9 & 10 ON and OFF variants will be conducted in a chinchilla model of OM to test the hypothesis that the observed phasevarion-mediated global changes in gene expression will provide a selective advantage in terms of niche adaptation in vivo. Aim 2 studies are designed to characterize the full complement of biological selective pressures that act on phasevarion switching in concert, within the mammalian host. Findings derived from Aim 1 studies will be validated in Aim 2 by comparison with expression profile analysis of key genes using samples archived from niche adaptation studies and via direct testing in the chinchilla model using NTHI reporter constructs combined with ex vivo imaging to monitor niche-specific gene expression.